Microbiological reprocessing of by-products of biodiesel production

ABSTRACT

An industrial method of reprocessing a mixture of by-products produced during biodiesel production containing a glycerol fraction and degumming residue as well as a feed additive, which may be obtained by this method.

The subject of the present invention is an industrial method ofreprocessing the glycerol fraction obtained during biodiesel production,as well as a new strain of Yarrowia lipolytica particularly suited foruse in this process.

The production of natural fuel oil components, so-called biodiesel,basically consists of the production of fatty acid esters from naturaltriglycerides, usu. plant lipids, via transestrification. U.S. Pat. No.2,271,619 reveals a method of converting the glycerides of higher fattyacids into esters of short-chain alcohols via the addition of amonohydroxyl aliphatic alcohol with less than five carbon atoms in thepresence of an essentially anhydrous alkali metal hydroxide as acatalyst. According to this patent, the process should be carried out ina reactor at a temperature of 86 to 212° F. (from 30 to 100° C.). Theamount of alcohol should not exceed 1.75 glycerol equivalents. Theamount of catalyst varies from 0.1 to 0.5% of glyceride by mass.

Subsequent patents amend or complement the patent described method. U.S.Pat. Nos. 2,360,844; 2,383,632; 2,383,580; 2,383,581; 2,383,614;2,383,633; 2,383,596 and 2,383,599 respectively describe consecutivevariants of the method revealed in U.S. Pat. No. 2,271,619 consistingof: a) the addition of an acid to the process and a spray-drying phase;b) distillation of unreacted alcohol; c) evaluation of the effects of acatalyst—suggestion of a pH range from 5 to 7; d) the use of a partialfatty acid ester technique; e) reclamation of unreacted alcohol andacidification of the liquid in order to improve the separation ofglycerol and esters; f) reprocessing of partially reacted glycerides viavarious methods; g) further addition of addition of methanol to amonohydroxyl aliphatic alcohol (mot methanol), in order to improveliquid phase separation; as well as h) the addition of a solvent inorder to improve phase separation.

Other patents suggest more extensive modifications and improvements.U.S. Pat. Nos. 2,494,366; 2,383,601; 3,963,699; 4,303,590; 4,371,470;4,668,439; 5,399,731; 5,434,279 and 5,525,126 are also largely based onU.S. Pat. No. 2,271,619. They are respectively related to: a) theaddition of an appropriate amount of acidic catalyst to the alkalinecatalyst; b) readdition of the acidic estrification catalyst; c)processing under constant temperature and pressure, from vacuum toatmospheric pressure, d) addition of a second alkaline catalysis stage;e) addition of a second estrification stage and the removal of the alkylester using an absorbent; f) introduction of a gaseous alcohol; g)carrying the reaction out at a lower temperature with additional acid;g) introduction of an improved phase separation method using an acid; aswell as h) the use of a catalyst consisting of a mixture of calciumacetate and barium acetate.

Independent of the technology used, the by-product of thetransestrification of glycerides in need of reprocessing is theso-called glycerol fraction, comprising soluble hydrophilic reactionproducts, meaning glycerol unused catalyst as well as remaining fattyacid esters and other reagents used during further stages of separationof transestrification products, i.e. phosphoric acid and inorganicsalts. In most industrial biodiesel production processes and/orby-product reprocessing used at present, the glycerol fraction is in theform of “glycerol water” containing glycerol at 20%-80%, as well as theremainders of the technological process such as soap (0-5%), fatty acidmethyl esters (0-5%), methanol (0-1%), monoglycerides (0-6%), ash (0-5%)and water to 100% volume.

Another by-product of biodiesel production, which is difficult toreprocess, is the so-called gum that is formed during the initialpurification and pH adjustment of plant or animal fats fortransestrification, encompassing the use of phosphoric acid. The arisingby-product chiefly contains phosphoric acid residues in conjunction withfats, proteins as well as other macromolecules present in the fats.Furthermore, this by-product contains free plant and animal fats atvarious concentrations from 0-10%, 0-10% protein, 0-5% ash, 0-1%glycerol as well as water.

The reclamation and/or reprocessing of by-products is a significantproblem in the production of biodiesel.

The goal of the present invention is to deliver a method of easilyreprocessing a mixture of the aforementioned by-products formed duringbiodiesel production. A particular goal of the present invention is todeliver an efficient method of obtaining easily absorbed biomass fromthese by-products, of high nutritive quality, which method could be usedindustrially in the reprocessing of the glycerol fraction and degumming,taking into account the varying composition of these fractions dependingon the technological process used for biodiesel production. The biomassproduced should be characterised by a high content of easily absorbedprotein and vitamins, as well as being suitable for use as a feedadditive.

Unexpectedly, such stated problems have been solved by the presentinvention.

The subject of the present invention is an industrial method ofreprocessing by-products obtained during biodiesel production,characterised in that yeast of the species Yarrowia lipolytica iscultured on a medium comprising an aqueous solution containing, as acarbon source, from 20.0 to 70.0 g/l of a mixture containing glycerolwater and gum from degumming, at a temperature below 34° C.,preferentially from about 28° C. to about 31° C., medium oxygen loadingof at least 20% saturation with O₂, a pH value maintained from 2.5 to7.5, until a substantial exhaustion of the carbon source available inthe medium, where, preferentially, the culture is maintained in aperiodical fashion, and a portion of the culture broth at the end of aproduction cycle is replaced with fresh medium. Preferentially, themedium contains at least one component selected from among from a groupencompassing: ammonium sulphate, potassium phosphate, magnesiumsulphate, urea, thiamine, sodium hydroxide, yeast extract, corn mash,Chitosan as well as Acepol, at rates of 0.5 to 15 g/L medium. Equallypreferentially the carbon source used is a mixture of glycerol water anddegumming residue, where the degumming residue comprises at least 15%.Preferentially, the culture is maintained at a pH from about 3.4 toabout 3.6, preferentially at 3.5±0.1, and culture completion isindicated by a pH increase to 4.5. The biomass obtained may bespray-dried, at a temperature of about 200° C. at the input and 90° C.at the tunnel egress. Preferentially, the culture is maintained in avolume of at least 1000 L. In a preferential embodiment, the drawn-offculture broth results in 15 to 35 g/L of dry yeast mass, biomassproduction occurs at a rate of 1.5 to 3.0 g/L·h or with an overallefficiency of 0.4 to 0.5 g dry biomass/g glycerol fraction added to themedium, whereas dry mass protein content varies from 30 to 50% by mass.The culture may make use of the Yarrowia lipolytica strain SKOTANdeposited in the IBPRS under the accession number KKP 2018 p.

The Yarrowia lipolytica SKOTAN strain has been deposited in the depositbank working in accordance with the treaty of Budapest and maintained bythe Instytut Biotechnologii Przemyslu Rolno-Spożywczego (henceforthIBPRS), ul. Rakowiecka 36, 02-532 Warszawa and has been given theaccession number KKP 2018 p. This is a wild-type strain, which has beenselected from among many strains of this species belonging to thecollection of the Uniwersytet Przyrodniczy of Wroclaw tested during theresearch on the present invention. The selection criteria chieflyconsisted of the culture conditions on a medium based on the glycerolfraction. First of all, with this strain of the yeast Yarrowialipolytica, it was possible to achieve a particularly preferable biomassproduction efficiency as well as a considerable tolerance of deleteriousculture conditions such as increasing osmotic pressure as well as arelatively low pH in the medium. Due to this, the culture process ismuch simpler since there is little risk of it becoming contaminated byother microorganisms. At the same time, the biomass produced possessespreferential nutritive quantities such as a high content of easilyabsorbed protein and vitamins, particularly of the family. Due to thisit can be used as a high-quality feed additive.

The subject of the present invention is also a feed yeast containingfrom 42% to 49.3% protein in dry mass. Preferentially, the total contentof the amino-acids Ile, Leu, Lys, Met, Cys, Phe, Tyr, Thr, Trp, and Valis over 36 g/100 g protein, preferentially from about 36.6 to about 36.8g/100 g protein. Equally preferentially, the content of the selectedamino-acids in the said protein is in the range defined in Table 3.

The next subject of the present invention is the use of the Yarrowialipolytica SKOTAN strain deposited at the IBPRS under the accessionnumber KKP 2018 p in the reprocessing of by-products of biodieselproduction, where the by-products comprise a mixture of glycerol waterand degumming residue. Preferentially, the biomass produced is used infeed production.

EXAMPLE 1

Basic medium composition (Medium 1) for the production of biomass of theyeast Yarrowia lipolytica on a medium based on the glycerol fractionfrom biodiesel production (g/litre):

glycerol fraction from 20.0 to 70.0, preferentially about 50.0 (NH₄)₂SO₄8.5-15.0, preferentially about 12.6 Urea 1.5-6.5, preferentially about4.0 MgSO₄ × 7H₂O 0.5-3.0, preferentially about 1.0 KH₂PO₄ 0.1-2.0,preferentially about 0.5 Yeast extract 0.1-2.0, preferentially about 0.5Tap water 1000 ml pH 2.5-7.5, preferentially about 3.5-4.0

The above proportions of medium components (Medium 1) should be weighedfor a volume of 1100 L and brought to 1000 L with tap water.

In certain cases, as appropriate to the production goal (i.e.contraction of the growth time, protein maximisation, optimalisation ofthe amounts and composition of amino-acids or dry mass, etc.) the mediumshould be supplemented with other components for enriching or regulatingthe process, such as those given as examples in Table 1 below:

TABLE 1 Compounds enriching the Effect on production medium time drymass amino-acid protein Ammonium sulphate − + + + Potassium phosphate− + + + Magnesium sulphate − + − − Urea − + + + Thiamine − + − − Sodiumhydroxide + + + + Yeast extract + + + Corn mash + + + + Chitosan + − − −Acepol + − − − Oxygen (from air) + + + + “+” denotes a positive effect“−” denotes a negative effect

The content of foreign mineral or biological substances (i.e. heavymetals, toxins, etc.) in the raw materials used in the production shouldnot exceed that allowed for feed products described in appropriate normsand regulations.

After completely dissolving the medium components, they are poured intothe bioreactor, and 100 L of cultured yeast cells are added from abioreactor with a 150 L working volume, cultured as above.

Culture conditions for various strains of the yeast Yarrowia lipolytica:

the culture should be maintained at a temperature of 25-35° C.(preferentially about 30° C.±1), at an agitation rate of 400-1200 RPM(preferentially about 700-800), an aeration rate of 0.2-4 L air/1 Lmedium/min. (preferentially about 1-1.5 L air/1 L medium/min.). The pHshould be maintained automatically using 10N NaOH. If copious foamingoccurs, a defoamer such as ACEPOL or another should be used.

Process control using a PH-STAT is based on the regulation of the pH ofthe feed yeast culture production medium. The regulation consists of themonitoring of culture medium pH during yeast production. The controlapparatus maintains a pH of 3.5 with oscillations from 3.4-3.6. Aninitial pH of 3.5 is an absolute requirement and is achieved via theaddition of sodium hydroxide. The stabilised pH level limits the growthof undesirable bacterial flora and makes it possible to obtain ahomogenous culture of Y. lipolytica without other yeasts and bacteria.

A pH increase above and beyond 4.5 means the termination of the yeastproduction process and is evidence of the exhaustion of all of theavailable nutrients in the medium.

The next significant culture parameter is the medium temperature. Thetemperature should not exceed 34° C. The optimal temperature of thereaction mixture is 28-31° C.

Culture oxygenation is also a significant parameter. Oxygenation shouldexceed 20% O₂ saturation. Such an oxygenation index may be obtainedthrough the use of a bioreactor with a FRINGS-type aerator (a FRINGSturbine). The oxygenation level affects the culture efficiency as wellas yeast cell morphology. Full oxygenation results in yeast as shownbelow in FIG. 1. Insufficient aeration of the medium results in myceliaof Y. lipolytica (FIG. 2).

The culture should be maintained until the exhaustion of the availablecarbon source (here glycerol) in the culture medium. 200 L of the cellsuspension should be left into which the medium components (Medium 1)should be weighed off for a total volume of 1100 litres, and 900 litresof water should be added. Such a culture method (periodic and repeated)may be carried out from 5 to 15 times.

This yeast propagation process makes it possible to obtain 15-35 g/L(preferentially about 33 g/L) of yeast dry mass at a rate of 1.5-3.0g/l·h (preferentially about 2.5 g/l⊕h), with an overall efficiency of atleast 0.4-0.5 g yeast dry mass/g glycerol fraction, (preferentiallyabout 0.60 g/g in the case of the Yarrowia lipolytica SKOTAN strain).

The dry yeast protein content varies from 30 to 50%. Using the Yarrowialipolytica SKOTAN strain, a yeast dry mass protein content of about 42%was achieved for the basic medium composition (Medium 1) describedabove, as well as from 42% to 48.6% for various additional mediumvariants described in Table 1 above.

Measurements of amino-acid content (expressed in grams per 100 gramsprotein) obtained from cultures of the Yarrowia lipolytica SKOTAN strainon various medium variants described above are shown in Table 2 below inconjunction with 1998 FAO-WHO requirements for reference proteins.

TABLE 2 Results for Human Chicken 1991 Y. lipolytica milk egg FAO/WHOSKOTAN Amino-acid protein protein reference Min Max Ile 2.9 5.6 2.8 2.383.43 Leu 5.8 6.4 6.6 4.0 5.77 Lys 4.2 5.0 5.8 4.14 7.48 Met + Cys 2.64.5 2.5 1.51 1.90 Phe + Tyr 4.5 7.5 6.3 8.9 10.4 Thr 2.7 3.2 3.4 3.724.91 Trp 1.1 1.0 1.1 0.83 0.96 Val 3.4 5.1 3.5 3.14 4.44 Totalamino-acid 27.2 38.3 32 27.9 39.3 OSERA index 100 63.7 81.7 EAA index100 61.4 80.8

The biomass produced may be further processed, such as through dryingusing known methods, in particular spray-drying, and then apportionedfor distribution and used as a high-quality feed additive, particularlyfor use in bovine and chicken feed. Condensation of the yeast suspensionfor spray drying was performed using the following methods: flocculationusing Chitosan, microfiltration, centrifugation in a filtrationcentrifuge. The condensation level depends on the type of spray-dryer.Drying should be performed at a temperature of 200° C. at the input and90° C. at the tunnel output.

The yeast is in powder form, with a specific smell and a beige-browncolour.

EXAMPLE 2 Production of Feed Yeast on a Medium Containing the GlycerolFraction (Glycerol Water) as Well as Degumming Residue

Production is carried out as in Example 1, with the difference thatinstead of the glycerol fraction, the medium was supplemented with ananalogous quantity of raw glycerol water and degumming residue atarbitrary proportions. Optimal mixtures contain degumming residue at 15%to less than 100% of the mentioned mixture added to the medium. The mostpreferential culture results were obtained using full aeration and a pHmaintained at 3.5±0.1 during culturing. Conditional to the use of such amixture is the use of an appropriate aeration level and method ofagitation in the reactor (the use of turbines instead of frame mixers).

The protein content in Yarrowia lipolytica dry mass varied from 42% to49.3% in multiple replicants, depending on the compounds added to enrichthe mixture, as listed in Table A. The results of amino-acid levelmeasurements (expressed in grams per 100 grams protein) obtained inyeast cultures of the Yarrowia lipolytica SKOTAN strain on variousmedium variants containing glycerol water and degumming residue is shownin Table 3 in conjunction with the 1998 FAO-WHO reference requirementsfor reference proteins.

TABLE 3 Results for Human FAO/WHO Y. lipolytica milk Chicken eggreference from Skotan Amino-acid protein protein 1991 Min Max Ile 2.95.6 2.8 2.89 3.48 Leu 5.8 6.4 6.6 4.61 5.85 Lys 4.2 5.0 5.8 4.61 5.54Met + Cys 2.6 4.5 2.5 2.27 2.76 Phe + Tyr 4.5 7.5 6.3 10.8 11.5 Thr 2.73.2 3.4 3.69 4.13 Trp 1.1 1.0 1.1 0.97 1.2 Val 3.4 5.1 3.5 3.81 4.41Total amino-acids 27.2 38.3 32 36.6 36.8 OSERA index 100 81.9 83.9 EAAindex 100 81.3 82.5

Yeast produced on glycerol-degumming residue mixtures change colour tobeige-pale brown and acquire a sharper smell as the degumming residuecontent is increased.

1. An industrial method of reprocessing by-products of biodieselproduction, characterised in that yeast of the species Yarrowialipolytica is cultured on a medium comprising an aqueous solutioncontaining, as a carbon source, from 20.0 to 70.0 g/l of a mixturecontaining glycerol water and degumming residue, at a temperature below34° C., preferentially from about 28° C. to about 31° C., mediumoxygenation in excess of 20% of O₂ saturation, a maintained pH of 2.5 to7.5, essentially until the exhaustion of the available carbon sourcecontained in the medium, where the culture is preferentially maintainedin a periodically repeated way, each time replacing a portion of thecultured broth with a fresh portion of medium following the end of aproduction cycle.
 2. A method according to claim 1, characterised inthat the medium additionally contains at least one component selectedfrom a group encompassing: ammonium sulphate, potassium phosphate,magnesium sulphate, urea, thiamine, sodium hydroxide, yeast extract,corn mash, Chitosan and/or Acepol, at a rate of 0.5 to 15 g/L medium. 3.A method according to claim 1, characterised in that the carbon sourceused consists of a mixture comprising glycerol water and degummingresidue, where the proportion of degumming residue is at least 15%.
 4. Amethod according to claim 1, characterised in that the culture ismaintained at a pH from about 3.4 to about 3.6, preferentially at3.5±0.1, and termination of the culture is signalised by a pH increaseto 4.5.
 5. A method according to claim 1, characterised in that thebiomass produced is spray-dried, at a temperature of about 200° C. atthe input and 90° C. at the tunnel output.
 6. A method according toclaim 1, characterised in that the culture is maintained in a volume ofabout 1000 litres.
 7. A method according to claim 1, characterised inthat 15 to 36 g/L of yeast dry mass is obtained from the separatedculture broth.
 8. A method according to claim 1, characterised in thatthe biomass production occurs at a rate of 1.5 to 3.0 g/L⊕h.
 9. A methodaccording to claim 1, characterised in that the biomass productionoccurs at an overall efficiency of 0.4 to 0.5 g dry biomass/g glycerolfraction added to the medium.
 10. A method according to claim 1,characterised in that the protein content in the dry biomass is from 30to 50% by mass.
 11. A method according to claim 1, characterised in thatw the culture makes use of the Yarrowia lipolytica SKOTAN straindeposited in the IBPRS under the accession number KKP 2018 p.
 12. A feedyeast containing from 42% to 49.3% protein in the dry mass.
 13. A feedyeast according to claim 12, characterised in that the said proteincontains the amino acids Be, Leu, Lys, Met, Cys, Phe, Tyr, Thr, Trp andVal at a level in excess of 36 g/100 g protein, preferentially fromabout 36.6 to about 36.8 g/100 g protein.
 14. A feed yeast according toclaim 12, characterised in that the content of the selected amino-acidsin the said protein is in the range defined in Table
 3. 15. A use of theYarrowia lipolytica SKOTAN strain deposited in the IBPRS under theaccession number KKP 2018 p in the reprocessing of by-products ofbiodiesel production, where the by-products comprise a mixturecontaining glycerol water and degumming residue.
 16. A use according toclaim 15, characterised in that the biomass produced is used as a feedadditive.